The neurotransmitter serotonin is a fundamental modulator of behavior, and serotonin deficiencies lead to numerous debilitating psychiatric conditions, including depression. The number of serotonergic neurons in the nervous system is relatively small, but they modulate the activities of enormous numbers of neurons. To accomplish this, serotonergic neurons send out long-range axonal projections that diffusely arborize in virtually every region of the brain, and elaborate an expansive neural network. However, the molecular and cellular mechanism underlying serotonergic circuitry formation is largely unknown. Recent studies have shown that the protocadherin (Pcdh) ? gene cluster plays an essential role in this process. The three mammalian Pcdh gene clusters (?, ?, ?) encode a large family of homophilic cell surface proteins that are stochastically expressed in random combinations in individual neurons. Genetic studies have shown that Pcdh? proteins mediate dendritic repulsion and self-avoidance in a manner that is similar to that of the fly Dscam1 proteins. We generated Pcdh? cluster deletion mice and found that they display depressive-like behaviors as well as disorganized serotonergic projections, most prominently in limbic structures that mediate emotional processing. The spatial distribution of serotonergic varicosities is randomized in the absence of Pcdh?, possibly due to a loss of repulsion between serotonergic axons. In addition, serotonergic axons in Pcdh? mutants are highly enriched in certain areas of the brain and depleted in others, suggesting that a guidance mechanism is also involved. Remarkably, conditional deletion of Pcdh? genes in serotonergic neurons recapitulates the serotonergic wiring phenotype as well as depressive-like behavior. In contrast, serotonergic wiring and affective function are unchanged when the deletion is restricted in the target areas. A working model for serotonergic axonal arborization is proposed based on these observations, whereby Pcdh? proteins in produced in serotonergic neurons mediate axonal repulsion, and this repulsive force counterbalances target derived guidance cues to define the diffuse distribution pattern of serotonergic axons, which is required for normal affective function The specific aims of this study are designed to rigorously test the validity of this working model and to further understand the underlying cellular and molecular mechanisms. In Aim 1, we will distinguish the roles of serotonergic neurons and target fields in serotonergic circuit formation, and determine the nature of the serotonergic wiring alterations. In Aim 2, we will investigate whether molecular diversity of Pcdh? proteins plays a role in this process, whether specific isoforms are required, and whether this role is Pcdh?-specific or replaceable by other Pcdh subclasses. In Aim 3, we will carry out single cell RNA-Seq to determine whether Pcdh isoform diversity exist in serotonergic neurons, and identify potential downstream targets mediating serotonergic axonal spacing by using Pcdh? deficient mice and appropriate controls. Knowledge gained from this study is expected to shed new light into circuitry mechanisms of depression and inspire novel therapeutics.